Protein characterization plays a central role in diverse areas of biological and biomedical research. Much of this characterization involves mass spectrometric analyses, capable of providing information on both the primary sequence and the post-translational modifications (PTMs) of a protein or its peptides. Knowledge of the primary sequence of a protein is important for establishing its identity, while knowledge of PTMs is crucial for understanding many aspects of its cellular functions.
Collision induced dissociation (CID) is a method commonly used in mass spectrometric analyses of proteins, being useful for fragmenting peptides prior to detection, ultimately allowing for their amino acid sequences to be elucidated, at least partially if not completely. PTMs can also be analyzed by CID, through various means, but the site of PTMs on peptides/proteins often cannot be determined. Complete sequence and PTM-site information for peptides often cannot be obtained through CID because of the slow-heating nature of its mechanism, in which peptide ions are gradually heated through multiple collisions with neutral gas species, with each collision adding to the internal energy of the peptide. The time-scale for these collisions is slow relative to the time for the induced vibrational energy of the peptide ion to be distributed throughout the molecule, and as a consequence weaker bonds are fragmented preferentially. Since not all bonds between amino acid residues are of the same strength, not all are broken, and so gaps arise in the sequence information obtainable by CID. Likewise, labile PTMs are frequently lost prior to dissociation of the main peptide backbone, so that fragment sections of the backbone no longer bear the PTMs, preventing localization of their sites on the peptide. Hence, even though CID has many favorable attributes, its nature inherently limits the information it can provide in structural characterization of peptides/proteins, prompting the development of alternate fragmentation methods.
Two new methods of peptide/protein fragmentation have recently been developed—electron capture dissociation (ECD) and electron transfer dissociation (ETD)—which can provide information complementary to that obtainable by CID. Significantly, both ECD and ETD induce fragmentation non-ergodically, i.e., without energy randomization in a molecule. This characteristic results in ECD and ETD inducing fragmentation essentially evenly along the peptide/protein backbone, independent of bond strength, providing more complete sequence information than CID. Further, labile PTMs are generally preserved during the fragmentation step, permitting the routine determination of their sites.
Electron capture dissociation (ECD) involves the reaction of electrons with gas-phase peptides or proteins having multiple positive charges (Zubarev, R. A., Kelleher, N. L., McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266; Cooper, H. J., Hakansson, K., Marshall, A. G. Mass Spectrometry Reviews 2005, 24, 201-222). Energy released during the capture of an electron by a multiply-charged peptide/protein ion may result in cleavage of its backbone, generally without disturbing its PTMs. In ECD, multiply-charged positive ions are normally formed by electrospray ionization (ESI) at atmospheric pressure, then transferred into the high vacuum of an FT-ICR mass analyzer where they are trapped, fragmented by reactions with electrons, and then detected. This original ECD method requires both positive ions and electrons to be simultaneously confined in the same spatial volume, generally requiring the use of a Penning ion trap, where confinement of charged particles relies upon the strong magnetic field provided by an expensive super-conducting magnet, an integral component of FT-ICR mass spectrometers. Consequently, though powerful, the original ECD method suffers from the substantial shortcoming of requiring highly expensive, specialized instrumentation.
Electron transfer dissociation (ETD) is very similar to ECD, but uses anions in place of electrons as the negative reagents (Syka, J. E. P., Coon, J. J., Schroeder, M. J., Shabanowitz, J., Hunt, D. F. Proc. Natl. Acad. Sci. USA 2004, 101, 9528-9533; Hunt, D. F., Coon, J. J., Syka, J. E. P., Marto, J. A. United States Patent Application Pub. No.: US2005/0199804A1). With ETD, generally, positively charged peptide/protein ions from an ESI source at atmospheric pressure are delivered into some form of electrodynamic ion trap in the vacuum system of a mass spectrometer, where they are confined and then reacted with anions from a supplemental ion source. The anions transfer electrons to peptide/protein ions and induce fragmentation in much the same manner as in direct ECD. A benefit of ETD relative to ECD is that it can be performed using relatively inexpensive quadrupole ion trap mass spectrometers, capable of simultaneously storing both positively charged peptide ions and anionic reagents (though not electrons) through the use of a dynamic electric field in place of a strong magnetic field. Like ECD, ETD has been demonstrated to be well-suited for providing information on the amino acid sequences of peptides, as well as the identity and site of labile PTMs. However, also like ECD, ETD suffers from the drawback of requiring expensive specialized mass spectrometers, which must include a means of simultaneously trapping both peptide ions and anionic reagents within the vacuum system of the mass analyzer, in addition to a supplemental means of anion production.
An alternate approach to performing ECD or ETD would be to react the peptide/protein ions with electrons or anions in the ion source of the mass spectrometer, at atmospheric pressure, outside the vacuum system of the mass analyzer. A practical method of performing in-source ECD or ETD at atmospheric pressure would have an advantage over conventional ECD/ETD methods in that dedicated mass spectrometers equipped with specialized ion trapping capabilities, as well as supplemental electron/anion production means, would not be required. All manner of mass analyzers, including those not originally designed for ECD/ETD experiments, could potentially be outfitted or retrofitted with an atmospheric pressure (AP) ECD/ETD source. Such a device would potentially make the powerful ECD/ETD technology more widely accessible, as researchers wishing to perform ECD/ETD on their peptide/protein samples would not need to acquire entire new instruments dedicated to the task.
To date, there have been two published reports of peptide ions being fragmented at atmospheric pressure through an ECD/ETD-like process (Delobel, A., Halgand, F., Laffranchise-Gosse, B., Snijders, H., Laprévote, O. Anal. Chem. 2003, 75, 5961-5968; Debois, D., Giuliani, A., Laprévote, O. J. Mass Spectrom. 2006, 41, 1554-1560). These reports came out of fundamental studies of fragmentation mechanisms active within a conventional PhotoSpray™ atmospheric pressure photoionization (APPI) source from AB Sciex (Concord, Ontario, Canada). The reports described how in the PhotoSpray™ source multiply-charged peptide ions from its pneumatic heated nebulizer may be transported by a flow of gas to a downstream photoionization region, where photoionization of an ionizable component of the gas produces photoelectrons, which may then be captured by the peptide ions and cause AP-ECD; ultimately, ions exiting the ion source—including the ECD products—are delivered through the atmosphere-vacuum interface of the mass spectrometer for mass analysis and detection. The significance of these early reports is that they served to demonstrate that ECD/ETD reaction products may be created at atmospheric pressure and then delivered intact into the vacuum system of the mass spectrometer; however, the researchers to first observe the phenomenon made no efforts to study or develop AP-ECD as a practical tool for peptide/protein structural characterization. This is attributable in part to the facts that the quality (general appearance and information content) of the AP-ECD spectra obtained were poor and that quantities of sample far in excess of those normally used in protein mass spectrometry were consumed to generate the spectra. Hence, it appears that the sensitivity of AP-ECD as originally demonstrated was too low for it to be recognized as a potential alternative to conventional ECD/ETD methods.
The low sensitivity of the original AP-ECD method is at least in part attributable to the nature of the means of peptide ion production, the heated nebulizer probe, designed and normally used to vaporize liquid sample streams bearing neutral analytes, to be ionized subsequently via separate means such as APPI. Since all the components of the heated nebulizer probe are at ground potential—including the internal pneumatic sprayer for nebulizing the liquid sample stream—there is no electric field in the nebulizer to promote charging of the liquid and thereby promote the formation of multiply-charged peptide/protein ions. The peptide ions that are generated directly from conventional heated nebulizers preexist in solution, and are liberated into the gas phase from droplets formed with a net charge during nebulization, as a result of statistical variations in the number of oppositely charged ions within each droplet. Droplet charging through random fluctuations in ion populations is a very inefficient process relative to the deliberate charging of the liquid via electrical means as is the norm in ESL At first glance, it may then appear a simple matter to increase the initial yield of peptide/protein ions for subsequent AP-ECD via photoelectrons, by replacing the grounded heated nebulizer of the APPI source with an ESI source. However, there have been no prior examples of electrifying the sprayer of a conventional heated nebulizer, to promote peptide/protein ionization, which would require substantial redesign and modification of existing hardware never intended to be electrified. Further, in one prior case where a dual-mode ion source coupling conventional ESI and APPI was used in the analysis of peptide/protein samples, when the APPI source was on, large suppressions of the ESI multiply-charged ions were observed, with no sign of ECD/ETD fragment ions (Syage, J. A., Hanold, K. A., Lynn, T. C., Horner, J. A., Thakur, R. A. J. Chromatogr. A 2004, 1050, 137-149). Altogether, then, the prior art surrounding AP-ECD does not suggest a straightforward means of increasing the sensitivity of the method to make it a viable alternative to regular ECD/ETD.
There has been one other prior mention of using an electron-based fragmentation method at atmospheric pressure as a tool for peptide/protein characterization (Whitehouse, C., White, T., Willoughby, R., Sheehan, E. United States Patent Application Pub. No.: US2006/0255261A1). In this case, an apparatus was envisioned in which the sprayed ions from two or more liquid inlet probes are mixed at atmospheric pressure, and in which one of the probes produces multiply-charged positive peptide/protein ions while another produces anionic reagents for ETD. Though on its surface such an apparatus may appear straightforward to implement and potentially viable, in practice there is a problem with the design which limits its efficiency, at least, and possibly prevents its successful operation altogether. The problem is that the two liquid inlet probes are situated in close proximity in an open spatial volume. Such a configuration is highly unfavorable for effecting ECD/ETD, as the strong electric field of the ESI probe used to generate positive ions will be experienced by the electrons/anions from the other probe, resulting in the negatively charged reagent ions being drawn towards the ESI probe, rather than towards the individual peptide/protein ions to be fragmented. This occurs because the electric field in the vicinity of the probe is much greater than that of individual positive ions. Negative reagents reaching the ESI probe will be neutralized there, possibly quenching the electrospray as a result of the accompanying voltage drop (due to the current from neutralizing negative charges), and definitely removing the negative reagents required for ETD. Though it may be possible to circumvent these problems by situating the two probes far apart, so that the ions from each meet in a region remote from the ESI source probe, where the electric field from the probe is diminished, this will inevitably result in poor transmission of ions into the reaction region and then into the mass analyzer. This is because positive ions initially follow divergent trajectories from the ESI probe and no means of guiding the ions to the reaction region has been included in the prior design. It is perhaps then no coincidence that no results have yet been presented for the multiple-probe AP-ETD source envisioned.
In summary, both ECD and ETD have been proven to be powerful fragmentation techniques for the mass spectrometric analysis of peptides/proteins, though each of these techniques require expensive, specialized equipment. An atmospheric pressure ECD/ETD method would have the advantages of requiring relatively less expensive hardware and would be suitable for use with all manner of mass analyzers, including those not expressly designed for ECD/ETD experiments. However, only a couple of AP-ECD/ETD methods have been reported, and none has been shown to be a viable alternative to conventional ECD/ETD techniques.